Various methods for the determination of protein in aqueous fluid have been reported in the literature. These methods include the Kjeldahl method, biuret method, Lowry method, dyestuff combination method, UV method and fluorometric method. Widely used methods for the assay of urinary protein are the Kingbury-Clark method; reported in J. Lab. Clin. Med., 11, 981 (1926); the Exton method; reported in Rinsho Byori, 15, 699 (1967); Merlemans method; reported in Clin. Chim. Acta, 5, 757 (1960) and the Coomassie brilliant blue method; reported in Anal. Biochem., 72, 248 (1976).
In general, protein interacts with various substances, particularly with dyes such as bromphenol blue, coomassie brilliant blue and eosine as well as metal ions such as silver (I), copper (II), zinc (II) and lead (II). Typically, the addition of protein to the reaction between a dye and a metal ion gives a spectral change to a dye-metal ion solution. Fujita et al report in Bunseki Kagaku Vol. 32, Pp. E379-E386 that the addition of protein to the reaction between pyrogallol red and molybdenum (VI) produces a different spectrum than that of the pyrogallol red-molybdenum (VI) complex solution. These authors report that among metal ions tested, large amounts of iron (II) interfered with the protein determination and that among anions tested, organic acids such as citrate, oxalate and tartrate ions decreased the absorbance at 600 nm. While large amounts of other ions did not interfere, Fujita et al report that large amounts of creatinine and amino acids caused a slight increase in absorbance at 600 nm.
Japanese Kokai Patent No. SHO 62[1987]-6170 discloses a test strip for protein determination comprising a molybdate salt, a pigment which forms a complex with molybdate and whose adsorption band is shifted in the presence of a protein and a chelating agent which combines with molybdate ions. A similar assay for trace amounts of protein is disclosed in Japanese Kokai Patent 61-155757 in which there is described the use of a chelating agent which is able to bond with molybdenum or a metal ion which is able to bond with oxalic acid, citric acid, phosphoric acid or their salts which are normally present in the test sample. This assay is a dye binding method using the complex of pyrogallol red and molybdenum. At low pH the dye-metal complex is red. The color changes to blue when deprotenated at higher pH. The protein causes the dye to deprotonate more easily (at a lower pH) by the interaction of positively charged amino acid groups stabilizing the negatively charged deprotonated dye-molybdate complex.
The major limitation to the transition metal-pyrogallol red method of protein detection is the interference of chelating agents and nitrogen compounds normally found in urine. Background color or the color without protein is also dependent on urinary interfering compounds. Citrate, phosphate, tartrate and oxalate shift the dye-metal complex to a red color (protonated form in Scheme I). ##STR2##
The extent of the shifts is given in Tables 1-3; the direction of the shift is given below in Table A. Presumably the carboxylic acid groups complex the metal to stabilize the red protenated form.
TABLE A ______________________________________ Affect on Blue Color Ion of Background ______________________________________ Ammonium No effect Oxalate Decreases relative to water Creatinine No effect Citrate Decreases relative to water Phosphate Decreases relative to water ______________________________________
Creatinine and amino acids such as glycine shift the complex to blue (deprotonated form). This effect is the same as that observed with protein. To observe this effect, the creatinine concentration must be higher than the range tested in Tables 1-3 which represents the physiological extremes in urine. These interferences have been reduced by the use of chelating agents or metal ions which do not react with the dye. Two Japanese patents to Wako Ltd. describe the use of certain chelates and metals to reduce interferences. These patents, 62[1987]-6170 and 61-155757 (1986) cite oxalic acid as the preferred species for limiting interferences associated with the use of molybdate-pyrogallol red reagents.
It has more recently been discovered that tungstate acts in a manner similar to molybdate in the presence of protein and a dye. Useful tungstate salts include sodium tungstate, potassium tungstate, lithium tungstate, ammonium tungstate or a tungstate with an alkyl, dialkyl, trialkyl or tetraalkylammonium ion or a phosphotungstate bearing a similar cation.